In the fields of foods, medical and pharmaceutical products, and the like, in order to ensure the quality and safety, the microbiological examination of products or raw materials is carried out. As for test methods thereof, official analytical methods are prescribed in the respective fields, and so far as the medical and pharmaceutical products are concerned, various testing methods for microorganisms are described in the Japanese Pharmacopoeia. Among these, as for nonsterile preparations and raw materials thereof, the microbial limit test is prescribed, and this includes microbial enumeration tests for measuring microbial counts and a test for the detection of specified microorganisms regarding specified pathogenic microorganisms and the like. Furthermore, as for crude drugs, the microbial limit test for crude drugs is prescribed in Chapter 5.02 of the tests for crude drugs of the general tests. However, in the case where the microbial limit test is applied to a Kampo extract preparation and a crude drug that is a raw material thereof, the growth of a target microorganism is inhibited due to the influence of antimicrobial substances (inclusive of antifungal substances, hereinafter the same) contained in the Kampo extract preparation and the crude drug, which becomes problematic for accurate detection of the target microorganism.
In general, the antimicrobial activity of a test sample may be removed by inactivating the antimicrobial activity (neutralization of growth inhibitors), increasing the dilution ratio of a sample liquid, increasing the amount of a medium, membrane filtration, and the like. However, a variety of antimicrobial substances are contained in a Kampo extract preparation and a crude drug that is a raw material thereof, or the like, and thus, it is extremely difficult to effectively inactivate these antimicrobial substances with a specified inactivating agent. For example, by adding lecithin and polysorbate to a medium in a known method for inactivation of antimicrobial substances, a high effect in the inactivation of paraben type or mercury type preservatives or the like is obtained, whereas, in many cases, a sufficient effect in the inactivation of Kampo extract preparations is not obtained. Actually, a crude drug that is a raw material of the Kampo medicine contains a variety of compounds ranging from low-molecular weight compounds to high-molecular weight compounds. The majority of these compounds are characteristic components in view of molecular structures (e.g., terpenoid, alkaloid, flavonoid, quinones, amino acids, fatty acids, sugars, etc.) and contain a lot of antimicrobial substances (e.g., terpenoid, alkaloid, flavonoid, tannins, etc.). A Kampo extract preparation is composed of several species of crude drugs, and thus, it contains a wide range of antimicrobial substances. Furthermore, the Kampo extract preparation contains a lot of insoluble fine particles resulting from a production method thereof, and therefore the membrane filtration which is widely used to remove antimicrobial substances cannot be employed. Meanwhile, increasing the dilution ratio or the amount of a medium can hardly be considered the method of choice, because it leads to a decline in the sensitivity.
In view of these problems, the present applicant has already proposed a method for removing antimicrobial substances in a test sample by adding activated carbon and acid clay or activated clay to a medium (PTL 1). This method is useful in the test for the detection of specified microorganisms; however, in the microbial enumeration test by the plate-count method using an agar medium, it is difficult to discriminate colonies because not only is the medium colored with the activated carbon, but also a degree of transparency of the medium is reduced by the acid clay or activated clay. Furthermore, it is not easy to separate and remove these substances, which leads to difficulties in accurately discriminating and measuring colonies.